: The removal of introns from messenger RNA precursors by splicing is a mandatory step in the expression of most genes of higher eukaryotes. Recent work has defined a rare class of introns in animal genomes with highly conserved splice site sequences. These introns begin with the dinucleotide AU and end with the dinucleotide AC as opposed to GU and AG which begin and end the sole previously identified class of introns. These AU-AC introns are spliced by an alternative spliceosomal mechanism involving at least one novel component: the U12 small nuclear RNA (snRNA). The U12 snRNA has been shown to interact by base pairing in vivo with the branch site sequence of an AU-AC class intron. The investigator proposes to investigate the role of the conserved nucleotides of the AU-AC splice sites by site directed mutagenesis and in vivo and in vitro splicing assays. Among the issues to be investigated are the role of the conserved residues in the multi-step spliceosome formation reaction and the two step splicing reaction; the communication between the 5' and 3' splice sites; the function of an apparent upstream splicing enhancer sequence; and the interactions of the two spliceosomal systems to allow proper splicing of AU-AC introns and adjacent GU-AG introns. The investigator will search for other novel components of the alternative spliceosomal pathway using affinity selection and crosslinking methods and the investigator will investigate the snRNA-pre-mRNA interactions in the alternative spliceosome. Suspected snRNA-pre-mRNA and snRNA-snRNA base pairing interactions will be tested in vivo by assaying for suppression of mutations in one RNA by compensatory mutations in the other RNA. Finally, the investigator will explore the phylogenetic distribution of this class of introns with particular emphasis on plants and lower eukaryotes to determine if AU-AC introns are as phylogenetically old as GU-AG introns. These investigations bear on the mechanisms involved in the expression of genes in health and disease and on fundamental questions concerning the origin and organization of modern genes.